Free Energy-Based Methods

to Understand Drug Resistance

Mutations

Elvis A. F. Martis and Evans C. Coutinho

Abstract In this chapter, we present an overview of various computational

methods, particularly, those that are used to compute the free energy of binding to
understand target site mutations that will enable us to foresee mutations that could
significantly affect drug binding. We begin by looking at the driving forces that lead
to drug resistance and throw some light on the various mechanisms by which drugs
can be rendered ineffective. Next, we studied molecular dynamic simulations and its
use to understand the thermodynamics of protein–ligand interactions. Building on
these fundamentals, we discuss various methods that are available to compute the
free energy binding, their mathematical formulations, the practical aspects of each
these methods and finally their use in understanding drug resistance.

Keywords Molecular dynamics Drug resistance MM-PB(GB)-SA




Free energy perturbation Linear interaction energy Computational mutational


scanning Thermodynamic integration

1 Drug Resistance Problem

Every organism attempts to survive in hostile conditions by making minor modi-

fications in its life cycle. Though these modifications are observed phenotypically,
genetic reshuffling and alterations are the underlying cause of these changes.
Although we are unable to accurately explain this phenomenon and its initiation, we
have been able to use this observed knowledge and empirically derive explanations
for such modifications. However, it may not always be necessary to know all the
details regarding genetic modifications, so long as we can correctly, at least
empirically, understand such observations, and put it to effective use to predict and
understand the drug resistance problem. Often the enzymes in the biochemical

E. A. F. Martis
E. C. Coutinho (&)

Molecular Simulations Group, Department of Pharmaceutical Chemistry,
Bombay College of Pharmacy, Kalina, Santacruz [E], Mumbai 400098, India
e-mail: evans.coutinho@bcp.edu.in

© Springer Nature Switzerland AG 2019 1

C. G. Mohan (ed.), Structural Bioinformatics: Applications in Preclinical Drug
Discovery Process, Challenges and Advances in Computational Chemistry
and Physics 27, https://doi.org/10.1007/978-3-030-05282-9_1
2 E. A. F. Martis and E. C. Coutinho

pathways undergo mutations to improve the survival rate of the organism by either
improving the protein function or catalytic efficiency and stability to escape the
inhibitory action of the drug. In the latter case, the motive for modifying the drug
target is to ensure that drug binding is weakened. Moreover, the mutations are such
that substrate binding is unaffected or minimally affected. Most of the computa-
tional methods employed to study the mechanism of drug resistance, attempt to
understand the differences in the binding patterns of the substrate and the drug
molecule, i.e. understanding the “substrate-envelope hypothesis”. Here, we pre-
sent an overview of those computational methods that employ free energy of
binding as a tool to gauge the differences in the binding of the substrate and the
drug molecule before and after mutation.
In the Sect. 1, we discuss the driving force for resistant mutations and throw
some light on the different mechanisms by which drug resistance can occur. In
Sect. 2, we present a brief overview of molecular dynamics, thermodynamics of
protein–ligand binding, and various methods for computing the free energy of
binding. The last section, Sect. 3, has a detailed discussion on various free
energy-based methods used to understand and predict the target site mutations
leading to loss in drug binding.

1.1 Overview of the Mechanisms of Drug Resistance

The drug-induced selection pressure [1–4] is the major driving force for infectious
organisms to try to evade the effects of drugs. One of the primary moves that any
organism will adopt is to disrupt the action of drug molecules by one or more
possible mechanisms. To show its effect, the drug must enter the cells and find its
target protein. As a primary defence mechanism against drugs, the organism may
down regulate the expression of influx channels that enable the entry of the drug,
resulting in a decreased concentration build-up within the cell. Another strategy that
hinders the build-up of the drug inside the cell is the upregulation of the expression
of efflux channels/pumps that facilitate the egress of the drug molecules. These
strategies are often very difficult to understand owing to the complicated pathways
involved in the upregulation or downregulation of various proteins associated in the
regulation of traffic to and from the cell. This attribute is difficult to study using
computational techniques that use free energy-based methods. Target site mutations
[5–8] that lead to disruption in the drug binding without significant loss of the
protein function [9, 10] is another mechanism of drug resistance. Such mutations
can be studied using computer simulations that enable us to estimate the free energy
difference between the drug binding to the mutant and the wild-type protein. An
essential factor to consider while understanding target site mutation is the fitness
cost associated with the mutational change. This can be estimated by the change in
the free energy of binding of the natural ligands/substrates; for example, a drop in
their binding energy indicates that substrate binding is impeded, which this leads to
increased fitness cost. This means the enzyme now must expend more energy to
Free Energy-Based Methods to Understand Drug Resistance Mutations 3

carry out the same reaction. Hence, we can assume that such mutations are seldom
seen, and if at all they occur, a compensatory mutation(s) will be seen to counter the
detrimental effects of those mutations [11, 12]. Another strategy adopted by
organisms is to increase the production of drug-metabolizing enzymes that modify
the drugs to their inactive form eventually leading to their elimination. A classic
example of this is the inactivation of penicillin by the enzyme b-lactamase.

1.2 Overview of Computational Methods to Study Drug

Resistance

Broadly, computer-assisted methods used to study drug resistance can be classified

into two categories based on the information they require and the output they return.
The first category of methods requires only 1D sequence data as input and the
output is generally a classification type, i.e. the test sequence is classified as a
resistant or a non-resistant sequence. Thus, the methods grouped under this class are
collectively called as “sequence-based” methods [13]. The workflow of these
methods is akin to machine learning or QSAR type classification methods. In a
nutshell, sequence-based methods require sequences with the corresponding bio-
logical activity data (Ki or IC50 or any other suitable numerical value) for the drug
under study. Such data can be curated from databases like HIVDB (for HIV
resistance, curated and maintained by Stanford University; [14, 15]) CancerDR (for
cancer resistance, curated by CSIR Institute of Microbial Technology and OSDD,
India; [16]), tuberculosis resistance mutation database (curated and maintained by
various departments and schools with Harvard University; [17], and many other
such databases. The data is then split into training and test sets to develop and
validate the predictive models. The advantage of such methods is that it is not
necessary to know the tertiary structure of the protein or the drug-receptor inter-
actions. Therefore, sequence-based methods are computationally inexpensive and
large amount of data can be trained to obtain decent quality predictive models in a
short time. However, they suffer from two major drawbacks; (1) a lot of a priori
information on drug-resistant mutations is needed to train/develop predictive
models and (2) no mechanistic insights or atomistic details can be obtained.
The drawbacks seen in the sequence-based methods are efficiently overcome by
structure-based methods [13, 18, 19]. Further, structure-based methods are the
methods of choice when atomistic details are desired. However, these additional
details come at an added computational cost and require high-resolution protein
structures to be able to make accurate and reliable predictions. However, unlike the
sequence-based methods, they do not require large a priori information on muta-
tions; on the contrary, they can be applied to systems where no data on mutation is
available. To assess the binding stability which is the basis for predictions, these
methods employ either empirical scoring functions that implicitly try to reflect the
free energy of binding or use techniques that compute the free energy of binding
4 E. A. F. Martis and E. C. Coutinho

per se. Molecular docking-based methods use empirical scoring functions to find
the best docking conformations, and these methods are computationally less
expensive. Therefore, they can be applied to assess many protein–ligand com-
plexes. The ligand can be docked to various mutant proteins to predict their binding
strength before and after mutations, and this will allow one to understand the effect
of the mutation on the binding strength. The accuracy of docking-based methods
relies on the accuracy of the scoring function, and they are best suited for rank
ordering of compounds rather than computing the absolute free energy of binding.
The major issue with docking-based methods is that most docking programs treat
proteins as rigid entities, and therefore, mutations in highly flexible protein–ligand
systems are poorly understood [19]. However, in recent times there have been
several attempts to incorporate protein flexibility in molecular docking [20]. This
has largely improved the enrichment scores. Due to the limited scope of this
chapter, such docking methods will not be discussed here and have been treated
elsewhere [21–25]. Molecular dynamics-based methods can incorporate flexibility
in the protein–ligand complexes, and in most cases, are the methods of choice as a
conformational sampling tool to explore the phase space accessible to the system
under study. The conformations sampled are used to compute the free energy
change. However, the drawback of MD-based methods is the computational cost,
which is several magnitudes higher compared to docking-based methods.
Another critical issue that must be addressed about the structure-based methods
is, how fast predictions can be made, in addition to how reliable are the predictions.
These methods find application in drug discovery programs, wherein additional
filters can be placed to weed out molecules likely to encounter a high level of
resistance or assist in suitably modifying leads to inhibit the mutant proteins. Drug
discovery itself is an extremely lengthy and expensive process, and an additional
filter like resistance should be economical in terms of time as well as money.
Moreover, such methods should also assist medicinal chemists during lead opti-
mization stages to identify potential groups that will help evade drug resistance and
avoid late-stage failures that lead to huge financial losses.

2 Molecular Dynamics Simulations and Free Energy

Calculations

2.1 Overview of MD and Conformational Sampling

Methods

Computer simulations are very useful in predicting changes in molecular properties

brought about by alterations in an atom or a group of atoms, particularly, amino
acid residues. Therefore, they find good application in predicting the effect of
mutations on drug binding at the active site or elsewhere. Protein design experi-
ments clarify the effect of a mutation on drug or substrate binding, thereby facili-
tating prediction of drug-resistant mutations. This way the program can be used to
Free Energy-Based Methods to Understand Drug Resistance Mutations 5

select all mutations wherein drug binding is hampered and substrate binding is
either improved or [26].
In case of free energy calculations, molecular dynamics (MD) simulations are the
most commonly used technique to generate conformational ensembles. Hence, it is
rightly called as one of the main toolkits for theoretically studying biological
molecules (Hansson et al. [27], Binder et al. [28]. MD calculates the time-dependent
behaviour of particles or atoms, by numerical integration of Newton’s second law of
motion and predicts the future positions and momenta. MD simulations have pro-
vided detailed information on the fluctuations and conformational changes of pro-
teins and nucleic acids upon drug/substrate binding. As a result, it is now routinely
used to investigate the structure, dynamics and thermodynamics of biological
molecules and their complexes. MD simulations have an advantage in that, starting
from an X-ray or NMR solved structure, it can provide insights into the dynamic
nature of biomolecules that are inaccessible to experiments. To accurately simulate
the behaviour of molecules, one must be able to account for the thermal fluctuations
and the environment-mediated interactions arising in diverse and complex systems
(e.g., a protein-binding site or bulk solution). This depends on how accurately the
force fields represent the atoms and treats the non-bonded interactions. A complete
account of force fields can be found in the review by Pissurlenkar et al. [29].
However, most of the biological events occur at timescales that are not routinely
reachable by classical MD simulations, for example, protein folding occurs in the
timescale of few seconds, whereas drug binding and unbinding occur in the time-
scale of few microseconds to milliseconds. The routine timescale that is feasible
using high-end servers equipped with graphic processing units [30–32] and dis-
tributed grid computing [33, 34], is few tens of microseconds, that is nearly 1/100th
of the timescale required to study protein folding. Conventional MD suffers from the
severe limitation that it is extremely difficult to sample high-energy regions and
surmount energy barriers, leading to inaccuracies in free energy calculations.
The limitations of classical MD simulations have motivated the development of
new conformational sampling algorithms that facilitate the sampling of confor-
mational space that is inaccessible to classical MD simulation. The simplest way to
encourage the system to sample the high-energy regions on the phase space is to
increase the target temperature [35]. This leads to increased kinetic energy of the
system that enables it to surmount these barriers. However, it has been argued by
many, that such elevated temperatures (*400 K and above) lead to physiologically
unrealistic states that may severely distort the results; however, such methods have
been found to be advantageous in improving the sampling efficiency during MD
simulations. Another method that uses elevated temperature to enhance the sam-
pling is the replica-exchange molecular dynamics (parallel tempering, [36, 37]). In
this approach, several replicas are simulated in parallel at different temperatures. At
appropriate intervals, the replicas switch temperatures with the nearest replica, and
this exchange is governed by the Metropolis acceptance criteria. However, all these
methods do not prohibit the system from revisiting the same conformational space.
This problem was resolved by adding the memory concept in molecular dynamics
6 E. A. F. Martis and E. C. Coutinho

(local elevation method [38] Metadynamics [39]) uses Gaussian potentials that
discourage the system from sampling the same conformational space. These are few
of the most commonly used methods to tackle sampling problems in molecular
dynamics, a complete account on enhanced sampling algorithms can be found
elsewhere [40–44].

2.2 An Overview of Thermodynamics of Protein–Ligand

Binding

Molecular interactions, between the ligand and receptor, are primarily non-covalent
in nature and governed by attractive and repulsive forces. In drug design experi-
ments, the goal is always to optimize the attractive interactions and reduce the
repulsive ones [45–47]. Moreover, these associations are temporary, and the
lifespan of such complexes are governed by the off rates (Koff) or the dissociation
constant (Kd), both of which indicate the binding strength of a ligand to its protein
counterpart. In the realm of thermodynamics, binding is governed by enthalpic and
entropic components [48] given by Eq. 1.

DG ¼ DH  TDS ð1Þ

where ΔG is the binding free energy; ΔH is enthalpy; ΔS is entropy and T is the

temperature in Kelvin.
The association is favourable, i.e. spontaneous when the ΔGGibbs is negative and
unfavourable otherwise. All the binding and pre-binding (recognition and
pre-organization) events in biomolecular associations are either enthalpy
(ΔH) driven or entropy (ΔS) driven. The enthalpic component represents several
types of non-covalent interactions like electrostatic, van der Waals, ionic, hydrogen
bonds and halogen bonds, while the entropic components reflect the contribution to
binding due the dynamics or flexibility of the system. Computing the enthalpic
component of binding has reached far heights, in terms of methods available for
calculating the aforementioned type of interactions. However, till date, calculation
of the entropic component is extremely difficult, and the algorithms are computa-
tionally very demanding.
The Gibbs equation is more relevant in biochemistry for calculating the free
energy and is given by Eq. 2:

DGGibbs ¼ RT ln Kd ð2Þ

where ΔGGibbs is Gibbs free energy, R is universal gas constant, T is the temperature
in Kelvin, Kd is the dissociation constant. Equations 1 and 2, along with the
Born–Haber cycle [46] (Fig. 1) form the basis for the development of the methods
used to compute the free energy binding. The two main methods are Free energy
perturbation (FEP) and Thermodynamics Integration (TI), both of which will be
Free Energy-Based Methods to Understand Drug Resistance Mutations 7

Fig. 1 Thermodynamic or Born–Haber cycle for the receptor-ligand binding

dealt with in the subsequent Sect. 2.3.2. However, measuring the dissociation
constants from simulations is a daunting task; nevertheless, computing the partition
functions from the molecular simulations is relatively easy. Hence, the ratios of the
partition functions can be used to estimate the free energy of binding, which is
given by Eq. 2a,

QPL
DG ¼ kB T ln ð2aÞ
QP QL

where kB is the Boltzmann constant, T is the temperature in Kelvin, Q is the

partition function with subscripts PL, P and L indicating protein–ligand complex,
protein, and ligand, respectively. This section presents a summary of thermody-
namics, which is imperative for understanding the application and methods
developed to compute binding free energy. More elaborate discussions on the
thermodynamics of protein–ligand binding can be found in the reviews by
Bronowska [48], and Homans [46].

2.3 Methods to Compute Free Energy Binding

Free energy is a quantity that can be measured for systems such as liquids or
flexible macromolecules with several minimum energy configurations separated by
high-energy barriers. However, its computation is far from trivial and the associated
quantities such as entropy and chemical potential are also difficult to calculate.
More so, the free energy cannot be accurately determined from classical molecular
8 E. A. F. Martis and E. C. Coutinho

dynamics or Monte Carlo simulations due to their inability to sample adequately

from the high-energy regions of the phase space, which also make important
contributions to the free energy. However, the free energy differences (DDG) are
rather simple to compute. The free energy binding for the non-covalent association
of two molecules (protein and ligand in this case) may be written as follows:


DGbind ¼ Gcomplex  Gprotein þ Gligand ð3Þ

The binding event is an additive interaction of many events [49–52], for example
solvation energy (Gsol), conformational energy (Gconf), energy due to interaction
with residues in the vicinity (Gint), and energy associated with different types of
motions (translational, rotational and vibrational, Gmotion). The classical binding
free energy equation now can be rewritten as follows:

DGbind ¼ Gsol þ Gconf þ Gint þ Gmotion ð4Þ

Directly computing the free energy from an MD or MC simulation is not trivial;

hence, the following methods have been formulated. Broadly, the methods used for
computing free energy are classified as partitioning-based methods or end-state free
energy methods and non-partitioning-based methods. The partitioning-based
methods partition the binding energy into various components as shown in
Eq. 4; however, this method has been highly criticized [53] stating that it is
physically unreal to partition the free energy into components.

2.3.1 End-State Free Energy Methods or Partitioning-Based Methods

The human body majorly comprises of water; hence, it is imperative to carefully

include the solvation effects while computing the free energy of binding. More
importantly, water plays a crucial role in ligand recognition and in the binding
phenomenon. In computational chemistry, the methods for incorporation of solvent
are divided into three groups: (i) continuum electrostatic methods/implicit solvent,
(ii) explicit solvent models with microscopic detail and (iii) hybrid approaches.
Historically, the continuum electrostatic methods were among the first to consider
the solvent effect, and they still represent very popular approaches to evaluate
solvation free energies, especially in quantum chemistry. Polarizable continuum
model (PCM, [54]), COnductor-like Screening MOdel (COSMO, [55]) and SMD
solvation model [56] are few popular models for treating solvent effects implicitly
in quantum chemistry. Continuum solvation methods are computationally eco-
nomical; however, the frictional drag of the solvent is highly underestimated and as
a consequence may drive the system to non-physical states. Moreover, solvent–
solvent and solute–solvent interactions are inadequately treated, posing a danger of
underestimating the effects of such interactions. The explicit treatment of solvent
enables one to consider the solvent–solvent and solute–solvent interactions. This
prohibits the systems from visiting non-physical states due to the inclusion of the
Free Energy-Based Methods to Understand Drug Resistance Mutations 9

dampening effect shown by the solvent atoms. The principal drawback of explicit
solvent models is the number of atoms to be considered in the system leading to
increased computational cost. However, with the help of GPU-based acceleration,
this drawback, now, is hardly any cause for worry.
The end-state free energy methods use the conformations extracted from an MD
or MC simulation, wherein the system is simulated by explicitly defining the sol-
vent. However, while solving the GB or PB equation, the solvent is implicitly
treated by defining the external dielectric constant for water (for most drug design
cases) and a suitable internal dielectric constant [57–61].

Molecular Mechanics-Poisson Boltzmann/Generalized Born Surface Area

(MM-PB/GB-SA)

The MM-GBSA [62–65] approach employs molecular mechanics-based energy

calculations and the generalized Born model to account for the solvation effects in
the calculation of the free energy. Similarly, the MM-PBSA [66–68] approach
solves the linear or nonlinear Poisson–Boltzmann equation [69–71], to account for
the solvation electrostatics, whereas the MM part is calculated as in MM-GBSA
from the derivative of the force field equations. Both these approaches are
parameterized such that they partition the energy components into various terms,
and the net free energy change is the sum of these individual terms (Coulomb, vdW,
solvation, etc.). MM-PBSA has gained considerable attention for estimating the
binding free energies of molecular complexes due to its exhaustive nature of
computing the solvation electrostatics by iteratively solving the PB equation,
whereas the GB method does not involve any rigorous and iterative procedure and
hence is faster. However, this does not necessarily guarantee that the MM-PBSA
method always outperforms MM-GBSA method. In MM-PB(GB)SA methods,
MD- or MC-derived conformational ensembles are used to compute the “average”
free energy of a state and this is approximated as follows:
 
hGi ¼ hEMM i þ GPBSA=GBSA  T hSMM i ð5Þ

where the angular bracket <> indicates average over the MD/MC conformations,
EMM is the molecular mechanics energy that typically includes bond, angle, torsion,
van der Waals, and electrostatic terms (see Eqs. 7c and 7d) and is evaluated with no
or extremely large (virtually infinite) non-bonded cut-off limit. The second term is
solved as mentioned in the preceding stanza and it forms the crux of this method.
The last term T <SMM>, is the solute entropy, which is estimated by quasi-harmonic
analysis [72, 73] of the trajectory or by normal mode analysis [74–76].
The following equation (Eq. 6) shows how the binding free energy is computed
from the energies of the ligand, protein, and its complex over all the MD or MC
snapshots. However, the snapshots can be obtained in two possible ways—one is
called the single trajectory approach and other is the multiple trajectory approach.
In the single trajectory approach, only the protein–ligand complex is simulated, and
10 E. A. F. Martis and E. C. Coutinho

the snapshots for the protein, ligand and the complex are extracted by defining
appropriate atom numbers from the parameter and coordinate file. However, in the
multiple trajectory approach, three separate simulations are performed, one each for
the protein, ligand and protein–ligand complex.
 
   
hDGbind i ¼ Gcomplex  Gprotein  Gligand ð6Þ

Furthermore, Eq. 1 is modified to accommodate solvation electrostatics and

hydrophobic terms as shown in Eq. 5. Here, Eqs. 7a–7d give the computation of
the individual terms,
DGbind ¼ DEMM þ DGsol  TDS ð7aÞ

DGsol ¼ DGsolelect þ DGnonpolar ð7bÞ

DEMM ¼ DEint þ DEelect þ DEvdW ð7cÞ

DEint ¼ DEbond þ DEangle þ DEtorsion ð7dÞ

Here, ΔEMM is computed in the gas phase using classical force fields, ΔGsol is
computed using PBSA or GBSA method, ΔGsol-elect is computed using PB or the
GB method, and the ΔGnonpolar is computed by the solvent accessible surface area
(SA). While employing the single trajectory approach, Eq. 7d generally cancels out
and hence makes negligible contribution to the binding energy.

Linear Interaction Energy (LIE)

Linear interaction energy [77–79] is similar to the MM-PB/GB-SA method with

regard to the partitioning of the electrostatic and van der Waals terms (polar and
non-polar contribution, respectively,); however, the use of the weighting parameter
for electrostatic and van der Waals interactions, is unique to this method. LIE
measures the binding energy by estimating the difference in the interaction energies
of the ligand in the solvent (unbound state) and in the protein environment (bound
state). Hence, to obtain these interactions, two separate MD simulations are per-
formed. In one simulation, only the ligand is placed in the solvent (mostly water) and
in the other, the protein–ligand complex is placed in the solvent. The formulation of
this method is based on deriving the linear response approximation from converged
ensemble interactions, most often extracted from well-equilibrated trajectories from
the MD simulation of the ligand with its surroundings (solvent or protein).
The mathematical formula for computing free energies using LIE method is
given in Eq. 8
 LS   LS    LS   LS  
DGbind ¼ a Ecoul PL
 Ecoul L
þ b EvdW PL
 EvdW L
ð8Þ
Free Energy-Based Methods to Understand Drug Resistance Mutations 11

LS
where the angular bracket <> indicates ensemble over the MD trajectory, Ecoul and
LS
EvdW are electrostatic and van der Waals interactions between the ligand and its
medium in the vicinity (PL—protein–ligand complex; L—ligand in solvent), and a
is the weighting parameter for electrostatic interactions, which is most often set to
0.5 [78]. This value is assumed due to the linear response of the surroundings to the
electrostatic field and was validated using more extensive computations on the ions
(Na+ and Ca2+) in water [80]. b is the weighting parameter for van der Waals
interactions and is set to 0.16−0.18 [81], which is a subject of much debate owing
to the difficulty in estimating the vdW’s contribution to the free energy of binding.
However, these values are obtained by empirical fitting the experimental binding
free energies. Moreover, the linear response of the vdW term is assumed by
observing the linear trend in the interaction of the hydrocarbons with the solvent
(water) that depends on the number of carbons in a hydrocarbon.

2.3.2 Non-partitioning-Based Methods

In non-partitioning methods, there is no partitioning of the free energy into various

components. Statistical mechanics plays a crucial role in deriving the relationship
between the free energy of a system and the ensemble average of the Hamiltonian
that describes the system. These methods are far more accurate than the previously
mentioned end-state free energy methods, but at the same time, are computationally
very demanding. Hence, while dealing with a large dataset of molecules against a
particular protein target, it is worthwhile to screen the molecules using a fast
method like high-throughput virtual screening [82, 83], followed by a flexible
docking-based screening, then use an end-state free energy method, and finally
employ the non-partitioning methods to study few tens of molecules. Here, we will
present a brief discussion on FEP and TI methods along with their mathematical
treatment, and then move on to explain the idea behind alchemical free energy
predictions.

Free Energy Perturbation (FEP) and Thermodynamic Integration (TI)

Most of the methods for free energy calculations are generally formulated in terms
of estimating the relative free energy differences, DG, between two equilibrium
states, or binding of two similar ligands to a common target. The free energy
difference between the two states I and II can be formally obtained by Zwanzig’s
formula [84, 85].

ðbDVÞ
DG ¼ GII  GI ¼ b1 ln eI ð9Þ

Here, b ¼ ðkB T Þ1

12 E. A. F. Martis and E. C. Coutinho

This represents a sampling of the differences in potentials (DV) of the two states
using Monte Carlo or molecular dynamics simulation over the potential of state I.
To ensure the convergence of these calculations, it is recommended that the
potentials of the two systems should thermodynamically overlap. For satisfying this
condition, correct conformations must be selected, which is a daunting task, and
hence, to achieve this, a multistep process is usually implemented. A path between
the states I and II is defined by introducing a set of intermediate potential energy
functions that are constructed as linear combinations of the initial (I) and final
(II) state potentials and these intermediate states are non-physical states (Eq. 10).
Vm ¼ ð1  km ÞVI  km VII ð10Þ

where the transition from one state to another is discretized into many points
(m = 1,…,n), each represented by a separate potential energy function that corre-
sponds to a given value of k, such that km varies from 0 to 1. Here, zero indicates
the pure initial state of the system and one indicates pure final state of the system.
The total free energy, thus, can be obtained by summing over the intermediate states
along the k variable.
X
n1
DG ¼ GII  GI ¼ b1 lnh½bðVm þ 1 Vm Þ im ð11Þ
m¼1

This approach is known as free energy perturbation (FEP) where Dkm = km−1 − km;
hence, it can be written as
X
n1
DG ¼ b1 lnhe½bDVDkm Þ im ð12Þ
m¼1

Since the potential difference can also be described as the derivative of the
potential with respect to km, Eq. 12 can also be written as,

X
n1
lnhe½b@km Dkm Þ im
@Vm
DG ¼ b1 ð13Þ
m¼1

Now, expansion of the Eq. 13 by the Taylor expansion series gives Eq. 14,

X
n1
he½b@km Dkm Þ im
@Vm
DG ¼ ð14Þ
m¼1

wherein 0 ! k can instead be written as an integral over k

Z1 @V ðkÞ
DG ¼ hb i dk ð15Þ
0
@k k
Free Energy-Based Methods to Understand Drug Resistance Mutations 13

Equation 15 is usually referred to as the thermodynamic integration (TI) method

for calculating the free energy change [86, 87]. In the early days of free energy
simulations, the TI approach was synonymous with the slow-growth method [88].
In the slow-growth method, the value of k is changed at each time step during the
MD simulation. While this method was claimed to be more efficient than the
discrete FEP formulation, nowadays, a “non-continuous” change in k is a better
choice (50–100 discrete points are usually recommended). This facilitates equili-
bration at each point, the addition of extra points at any time, and use of any pattern
of spacing between the k-points, to optimize the efficiency.

Alchemical Free Energy Perturbation

Here, the free energy is computed by transforming a molecule from one state
(bound-solvated) to another state (unbound-solvated) through several physically
unrealistic states, that are called as alchemical states, hence the name “Alchemical
Free energy” [89, 90]. This method is regarded as one of the apt methods to study

Fig. 2 Thermodynamics cycle for computing alchemical free energy binding. Image reproduced
from Wang et al. [91] [open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY)]
14 E. A. F. Martis and E. C. Coutinho

the effect of mutations on the drug binding affinity (Fig. 2). The total free energy
change in a thermodynamic cycle in any alchemical transformation is equal to zero.

DG1  DG4  ðDG2  DG3 Þ ¼ 0 ð16aÞ

DG1  DG4 ¼ DG2  DG3 ð16bÞ

3 Application of Computational Methods to Understand

Drug-Resistant Mutations

3.1 Computational Mutation Scanning

Computational mutation scanning [92] is a useful method to explore the sensitivity

to changes in the composition of the amino acid in a protein-binding site (Fig. 3). In
computational mutation scanning, the wild-type amino acid residue is mutated to
another amino acid in the binding pocket or elsewhere. However, the most widely
practised method is to mutate any amino acid residue to an alanine, since it is the
simplest amino acid with a side chain (not glycine because it is devoid of a side
chain). Hence, this method is equivalent to the experimental “alanine-scanning
mutagenesis”, which is a powerful tool to investigate and confirm the important
interactions in the protein–protein interface and protein–ligand interactions. In
computational alanine scanning, all atoms from the Cb carbon atom of the amino
acid under study are replaced by three hydrogen atoms to convert it to an alanine.
After the mutation, the change in the binding energy is estimated either using
docking with an appropriate scoring function or by MM-PBSA or MM-GBSA to
compute DDG (Eq. 17c). By scanning with alanine at various positions in the
binding cavity, important residues can be identified, as mutating an important
amino acid will drastically decrease the binding energy.

DGWild
bind ¼ DGcomplex  DGreceptor  DGligand
Wild Wild
ð17aÞ

bind ¼ DGcomplex  DGreceptor  DGligand

DGMut Mut Mut
ð17bÞ
h i h i
DDG ¼ DGMut
bind  DG Wild
bind ¼ DGMut
complex  DG Wild
complex  DG Mut
receptor  DG Wild
receptor

ð17cÞ

In the context of predicting drug-resistant mutations, one must perform alanine

scanning in the binding site on two complexes, i.e. with the substrate bound complex
and the inhibitor-bound complex. The change in the binding energy after mutation is
computed for both the systems, viz., for inhibitor and the substrate. A decrease in the
binding affinity for the inhibitor with negligible or no change in the binding affinity
for the substrate indicates a hotspot amenable to resistant mutation, these spots are
Free Energy-Based Methods to Understand Drug Resistance Mutations 15

Fig. 3 Thermodynamic cycle for computing free energy change between mutated and wild-type
protein

termed as “mutational hotspots”. The method follows the substrate-envelope

hypothesis [93–95], which states that there is a large fitness cost that needs to be paid
if one mutates an amino acid residue that is involved in substrate binding. Mutating
such amino acids could lead to impaired enzyme function resulting in the death of an
organism. This can be put to appropriate use by developing inhibitors that com-
pletely overlap in the substrate binding region, leading to a lower predisposition
towards developing drug resistance [96–99].
16 E. A. F. Martis and E. C. Coutinho

However, a major drawback in alanine scanning is that when mutating a large

amino acid residue to alanine one can only study the effect of decreasing the side
chain or loss of charged groups in the binding site. It is difficult to understand the
resistant mutation, wherein there is a change in charged amino acid residue, for
example, arginine replacing aspartate or a large amino acid replaces a small amino
acid residue. Nevertheless, computational alanine scanning has been successfully
used to predict mutational hotspots.
Hao et al. [100] reported a modification of computational alanine scanning
(CAS), named computational mutation scanning (CMS) to study drug resistance in
six HIV-1 protease inhibitors. This protocol is an improvised version of the clas-
sical CAS that enables a geometry optimization step and incorporates entropy
calculations by means of normal model analysis. Using a single trajectory approach
and modifying the standard MM-PBSA protocol, to allow for mutations with other
amino acid residues, they computed the change in the binding affinities (DDG) of 77
drug-mutant combinations (includes single and double mutants). They obtained
promising results with *83% consistency with the experimental observations,
demonstrating that the prowess of the method lies in identifying the binding hot-
spots. However, Hao et al., do not report the change in the binding affinity for
various substrates, from which they could have investigated the substrate-envelope
hypothesis for the HIV-1 protease. This could have led to interesting findings
facilitating our understanding about those mutations that would lead to a decrease in
the enzyme function, either leading to the death of the organism or compelling a
compensatory mutation to counter the lethal effects of any mutation. This infor-
mation can be used to unravel the role and need for double, triple or even multiple
mutations.
Tse and Verkhivker [101] used CAS along with residue interaction network to
elucidate the effects of inhibitor binding on the network of residues in ABL kinase.
They showed the utility of this combination in deducing the critical networks of
amino acid residues and the changes that follow upon inhibitor binding, using a
selective kinase inhibitor (nilotinib) and two promiscuous (bosutinib and dasatinib)
kinase inhibitors. The changes in the interaction networks in the enzyme holds key
hints to unravel the mystery of how drug-resistant mutations are seen for ABL
kinase inhibitors. Moreover, the mutations that occur far from the binding site can
also be explained, since a mutation far off from the site can affect drug binding
through a cascade of events that eventually percolate into the binding site through
the changes in the residue interaction network. CAS followed by MM-PBSA added
the energetic component to locate the hotspots that could lead to drug resistance in
the kinase inhibitors

3.2 MM-PB(GB)-SA

MM-GBSA or MM-PBSA are two widely used free energy methods employed to
understand the effects of mutations on the drug binding affinity, moreover, these
Free Energy-Based Methods to Understand Drug Resistance Mutations 17

methods are successful in predicting likely mutations leading to drug resistance.

These methods are able to predict due to their amenability to decompose the free
energy into its components at the residue level that leads to better understanding of
the effect of mutations on drug binding. Lethal effects of the V82F/I84V double
mutation in HIV-1 protease on amprenavir were demonstrated using MM-PBSA
approach on snapshots obtained from the well-equilibrated protein–ligand complex
[102]. It was reported that amprenavir lost its binding affinity due to distortions in
the binding site, hence weakening many favourable interactions (DDG = 3.73 kcal/
mol). Such a distortion of the binding site was previously observed and attributed to
the rapid flap movements seen in this double mutant which is absent in the
wild-type HIV-1 protease [103]. Furthermore, newer inhibitors, that are very close
structural analogues of amprenavir, like TMC126 (DDG = 2.01 kcal/mol) and
TMC114 (darunavir, DDG = 3.45 kcal/mol) were also seen to be affected by these
mutations, though to a lesser extent than amprenavir. Despite structural distortions
in the binding site, it had no effect on the substrate binding, and hence, the catalytic
process was unhindered.
Hou et al. [104] combined MM-GBSA with the positional variability approach,
to modify Kollman’s FV value [105] to give a new scoring function also called FV
(Free energy/Variability) score. Using the FV score, they evaluated the binding of
six substrates that are hydrolysed by HIV-1 protease and confirmed Kollman’s
[105] observation that drug-resistant mutations are more likely to occur at less
conserved regions. The FV score reported by Hou et al. comprises two components,
one that reflects the binding energetics at the per-residue level, obtained by
MM-GBSA, and the second component is the sequence variability that represents
the conservation of amino acids at each position. Using this score, one can identify
amino acid residues that are crucial for substrate and inhibitor binding, and thus
classify the residues that are exclusively involved in substrate binding and those
that are exclusive for inhibitor binding. Such a classification when coupled with the
positional variability of amino acid residues can extract those positions with low
conservation and exclusivity for inhibitor binding; such positions are highly
amenable to mutations leading to drug resistance. Employing this method Hou et al.
confirmed their previous observation [102] that the V82F/I84V double mutations
are lethal for many FDA approved HIV-1 protease inhibitors, whereas TMC126 is
still active against this mutant.

3.3 Vitality Analysis

One of the primary drawbacks of the aforementioned methods to predict

drug-resistant mutations is their inability to accurately estimate the binding affinity
for the substrate molecule(s). The fitness cost of the mutation can be estimated by
gauging the change in the binding affinity of the substrate to its enzyme target; any
perturbation in the substrate binding is likely to affect the function of the enzyme.
Therefore, computing the catalytic efficiency of the enzyme before and after
18 E. A. F. Martis and E. C. Coutinho

mutation will enable us to understand the fitness cost. Pioneering work in this line
was done by Gulnik et al. [1]. In this work, they have determined the catalytic
efficiency (Eq. 18a) of HIV-1 protease following few active and non-active site
mutations. This principle was incorporated in terms of free energy change by
Warshel et al., and they employed this method (Eq. 18b) to computationally predict
the likely mutations that could potentially abolish drug binding leading to drug
resistance. This method is aptly named as “Vitality approach” wherein higher
vitality values indicate that the resistance is more likely as there is little chance of
increase in the catalytic efficiency of the enzyme. The basic workflow adopted by
Warshel et al. [106, 107] is to estimate the change in the drug binding before and
after mutation, depicted in the first part of Eq. 18b and then estimate the catalytic
efficiency by determining the binding of the substrate by modelling the transition
state (TS) conformation of the enzyme, depicted in the second part of Eq. 18b.
However, the challenge of employing this method to predict likely mutations is that
a thorough knowledge of the catalytic mechanism of the enzyme is essential.
Nonetheless, this method is far more accurate and truly predictive in nature. This is
exemplified by the fact that Warshel et al. successfully used this method on six
clinical agents active against HIV-1 protease.

Ki kcat
Km mutant
Vitality value ¼  ð18aÞ
Ki kcat
Km WT

cM 1

ln ffi DDGbind
N!M
ðdrugÞ  DDGbind
N!M
ðTSÞ ð18bÞ
cN RT

where Ki = inhibition constant; kcat = constant that defines the turnover rate of an
enzyme-substrate complex to the product; Km = Michaelis constant.

4 Concluding Remarks

This chapter describes important computational methods that have been proven
extremely helpful in gaining insights into mutations leading to drug resistance. We
have attempted to introduce methods used to compute the free energy of binding
along with their mathematical formulations, practical implementation and pros and
cons of such methods. Finally, we have discussed a few applications of such
methods to study drug resistance.

Acknowledgements E. A. F. Martis and E. C. Coutinho are grateful to Ian R. Craig, Ph.D.

(BASF, Ludwigshafen) for his critical comments and feedback on this chapter. The authors are
grateful to Department of Science and Technology (DST), Department of Biotechnology
(DBT) and Council of Scientific and Industrial Research (CSIR) for their financial support to build
the High-Performance Computing system at the Department of Pharmaceutical Chemistry,
Free Energy-Based Methods to Understand Drug Resistance Mutations 19

Bombay College of Pharmacy. E. A. F. Martis and E. C. Coutinho are also thankful to nVIDIA
Corporation for their hardware support grant. E. A. F. Martis is indebted to BASF, Ludwigshafen,
Germany for the Ph.D. fellowship and the MCBR4 (2015) consortium (Prof. Dr. P. Comba,
University of Heidelberg; Prof. Dr H. Zipse LMU, Munich and Prof. Dr. G. N. Sastry, IICT,
Hyderabad for MCBR visiting fellowship to Heine-Heinrich University of Düsseldorf, Germany).
E. A. F. Martis would also like to thank Prof. Dr. Holger Gohlke, Heine-Heinrich University of
Düsseldorf for his guidance during the sabbatical in his CPCLab. Gratitude is expressed to
Sandhya Subash, Ph.D. (Bristol-Meyer-Squibb, India), for her assistance in preparing and
proofreading the drafts of this manuscript.

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